Project Abstract The proposed project aims to create a new weapon in the arsenal of the CDC, for use in the `winnable battle' for improved food safety: a low-cost, hand-held, high-sensitivity, rapid-sensing platform to detect pathogens and biological and chemical toxins in the field, such as on farms, at food processing plants, and in medical facilities. This sensor device incorporates a new class of supercharged binding proteins, created through newly developed biodesign technologies, onto a sensor chip with a metastructured gold film, to produce a one- thousandfold enhancement in the surface plasmon resonance (SPR) shift upon binding of the target pathogen. Other than the engineered binding protein, the overall device uses only low-cost disposable detector chips fabricated via standard semiconductor fabrication processes, and off-the-shelf optical components, arranged in a more compact and robust geometry than any other known commercially available SPR detector. This research will initially target the detection of Listeria monocytogenes serotype 4b, which is responsible for an estimated 260 deaths and $2.8 billion in outbreak containment costs each year. The broader underlying principles can be applied to tap into the rapidly growing In Vitro Diagnostics (IVD) market (valued globally at $50 billion in 2014), to achieve the long-term goal of the rapid, sensitive, highly specific detection of most viral and gram-negative bacterial pathogens which are relevant to public health, both domestically and abroad. The proposed research will proceed with 4 specific aims:1) Design and optimize short-chain variable fragment (ScFv) proteins, based upon wild-type monoclonal antibodies to L. monocytogenes serotype 4b (the target pathogen) 2) Supercharge the designed ScFv protein by mutating antibody residues not essential for target binding, 3) Use standard semiconductor fabrication techniques to fabricate a gold plasmonic metasurface on a transparent fused silica substrate, and attach the designed ScFv protein to it using standard gold-thiol and biotin-streptavidin chemistries, and 4) Measure the SPR resonance shift as ScFv binds to its target (ie Listeria.) As a proof of concept of the highest-risk, highest reward component of the device (i.e. development of the supercharged binding protein), a model heme-binding protein named Mega(-) was designed, synthesized, and characterized. Mega(-) demonstrated a large, binding-induced conformational change, which caused a refractive index change, which is calculated to correspond to a 100?1000fold enhancement in SPR signal, relative to commercially available ligand?antibody detection schemes. Structural elements of Mega(-) were shown to undergo a phase transition from an ensemble of random-coil states into a more restricted ensemble of structured states, by tuning solution properties (and thus the range of electrostatic effects in the structure) with or without the presence of ligand. Thus, the designed binding interaction was shown to be largely insensitive to folding/unfolding phase equilibria?two features essential to SPR signal enhancement.